Cell-filled device of modifield cross-section hollow fiber membrane type

ABSTRACT

To provide a cell-filled device that is suitable for use in, for example, an implantable or circulation type hybrid artificial organ. In a cell-filled device including hollow fiber membranes whose hollow portions are filled with cells, the hollow fiber membranes have modified cross sections, and a cell aggregate provided in each of the hollow portions has cells formed into two or more layers in arbitrary directions, provided that the distance from an arbitrary point of the cell aggregate to the nearest inner wall of hollow fiber membrane is less than 75 μm. This cell-filled device enables effective use of cells without the necrosis thereof. Further, according to the present invention, there is provided a method of manufacturing the cell-filled device.

TECHNICAL FIELD

The present invention relates to a cell aggregate obtained by fillingcells in modified cross-section hollow fibers, and the use andmanufacturing method thereof. A cell-filled device of a modifiedcross-section hollow fiber membrane type of the present invention issuitably used in various applications such as an implantable orcirculation type hybrid artificial organ, material production devices(e.g., bioreactors), and cell incubators (stem-cell amplifiers).

BACKGROUND ART

In recent years, as approaches for treating organs or tissues havingfunctional failure or functional defects, the development of hybrid-typeartificial organs (also referred to as biological artificial organs),and regenerative medical technologies, in which cultured cells andbiocompatible materials are combined, have received much attention.

Currently, for instance, not less than 600,000 people are allegedlysuffering from liver diseases in our country. In addition, about 50,000patients have died a year because of liver diseases. Of those, about1,000 patient deaths are due to acute liver failure and the remainderthereof is due to chronic hepatic insufficiency including hepatoma. Abasic therapy for liver diseases such as hepatic insufficiency is livertransplantation. However, there is a large problem in that there is aninsufficient number of offerers who are willing to donate their organs(i.e., donors). Therefore, the development of artificial livers has beendemanded.

However, it is difficult to replace a total of 500 or more complicatedliver functions with only an artificial means. As to an artificialliver, recently, a biological artificial liver using hepatic cellsthemselves has received much attention.

For the biological artificial liver, which is a representative exampleof a hybrid artificial organ, an extracorporeal circulation typetherapeutic system is in the mainstream. The biological artificial livercarries out a therapeutic treatment by allowing a substance exchangethrough a plasma separator between a circuit on the body side fordrawing out blood from all of the hepatic failure patients andcirculating the blood and a circuit on the artificial liver module'sside for carrying out a plasma circulation to metabolize and detoxifythe plasma on the artificial liver module's side.

For such an artificial organ module, using dispersed cells isinsufficient. That is, monolayer cultures which have been conventionallyused for incubating cells cannot avoid loss or decrease of cellularfunctions. Thus, it is important to establish and use a multicellularaggregate body that resembles a living tissue.

From such a point of view, recently, a method of culturing an organ-likeaggregate such as a spherical cell aggregate (spheroid) or a cylindricalcell aggregate (cylindroid) has been newly established, so that the highfunctional expression and long-term functional maintenance of cells willnow be possible.

For example, as a method of culturing a spherical aggregate (spheroid),the inventors of the present invention have developed a method offorming a spheroid in a polymer base material such as apolyurethane foam(PUF) (JP10-29951A; and H. Ijima et al., “Tissue Engineering”, Vol. 4,No. 2, p. 213-226 (1998)). The PUF is made of a porous material having amain framework and a thin membrane beam structure. In addition, acertain degree of passage is formed between the pores of PUF, so a highdensity culture can be achieved under a good environment for substanceexchange. When hepatic cells are cultured in the pores of PUF, about 200hepatic cells gradually aggregate together to form many sphericalmulticellular aggregates (spheroids) each having a diameter of about 100μm spontaneously. The inventors have succeeded in developing ashort-term application type (about 10 days) biological artificial liveron a human clinical scale by means of a spherical aggregate (spheroid)using this culture method.

Furthermore, the inventors of the present invention have found thathepatic cells can be introduced into hollow fibers in a very dense stateby means of a centrifugal force as a result of seeking out a compactartificial liver of a long-term application type. They have finallyobtained an artificial module having a cell density of 2.4×10⁷ cells/cm³per module (see “Abstracts of 31st Summer Seminar Lectures in 2000 ofSociety of Fiber Science and Technology, Japan”, p. 115-118).

Furthermore, when an improvement in operability of the artificial liverat bedside and resolving the chronic lack of donors are taken intoconsideration, there is a need of a more compact artificial livercapable of maintaining its functions for a long time. Therefore, theinventors of the present invention have developed a higher density ofhepatic cell aggregate (hepatic cell organoid) (JP 2002-247978 A).

However, those conventional cell aggregates (organoids) have beenlimited to spherical one(spheroid) or cylindrical ones (cylindroids)using perfect circle-shaped hollow fiber membranes. Of those, the cellaggregate of a hollow fiber membrane type is excellent in its handlingproperties or functionality as a device. On the other hand, if the innerdiameter of the hollow fiber membrane used is too large, sufficientamounts of oxygen and nutrients would not diffuse to the cells locatedat the center of the cell aggregate, causing the necrosis thereof. As aresult, there were problems in that one would not be able to use thecells being filled in the hollow fiber membrane efficiently withoutwaste. Utilization efficiency of the cells becomes an extremely largeproblem when a cell source for making the cell aggregate (organoid) israrely available like one of a brain-death donor origin. In contrast, ifthe inner diameter of the hollow fiber membrane is too small, themanufacture of uniform hollow fiber membranes and the modularizationthereof becomes difficult. Furthermore, an airlock or the like can becaused and sometimes affected the operation of uniformly filling thecells.

On the other hand, when attention is paid to the structural aspects as asubstance production device, a cell culturing device of a hollow fibermembrane type is known and can be roughly classified into a type ofculture cells on the internal side of a hollow fiber membrane andsupplying a culture medium to the external side thereof and the oppositetype. In those cases, in general, a perfect circle-shaped hollow fibermembrane has been used, but sometimes other hollow fiber membraneshaving a different shape than a perfect circle has been used. Forinstance, JP 62-171678 A discloses that cells are incubated inside oroutside a modified hollow fiber membrane having a fine extending in alongitudinal direction from the outer peripheral portion thereof. Inaddition, JP 63-233777 A discloses that cells are incubated outside thehollow fiber membrane having unevenness in a hollow portion.

The former case has a description that the hollow fiber membrane may beoval instead of a perfect circle. However, it was the hollow fibermembrane that requires a fin (finny protrusion) on the external portionof the membrane. A main purpose of the fin is to prevent close contactbetween the membranes to improve the dispersibility of a culture mediumand cells. In the latter case, furthermore, the inner portion of themembrane was formed with irregularity in the longitudinal direction toprevent clogging of the membrane by causing turbulence in a culturemedium flowing in the membrane.

As a complex, furthermore, U.S. Pat. No. 5,015,585 discloses a hollowfiber membrane having a so-called double structure in which anotherhollow fiber membrane is incorporated in a hollow fiber membrane. Inthis case, a gap between two hollow fiber membranes is made uniform forthe purpose of keeping a survival rate of the cells filled in the gap.However, in obtaining the double-structured hollow fiber membrane, it isvery difficult to make the structure uniform unless the raw materials ofthe membrane, physical properties of the membrane, size of the module,and the like are limited. Besides, in this case, the cells cannot beexpected to be filled uniformly. In terms of this fact, to a largeextent, it was not practical.

In this way, various studies have been made on the shapes of cellaggregates and the hollow fiber membrane type cell culturing devices.However, no technology has attempted to increase the efficiency of usingthe individual cells by fully working on the cross-sectional shape anddiameter of the membrane on the basis of the cell aggregate formed inthe hollow fiber membrane.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a cell-filled device ofa modified cross-section hollow fiber membrane type, in which a cellaggregate is formed by filling cells in a hollow fiber, allowingefficient use of the filled cells without waste to enable an increase inutilization efficiency of cells. Another object of the present inventionis to provide an artificial organ that utilizes such a cell-filleddevice of a modified cross-section hollow fiber membrane type. Stillanother object of the present invention is to provide methods ofmanufacturing the above device and the artificial organ.

As a result of concentrated study, the inventors of the presentinvention have completed the present invention by finding out that acell aggregate (organoid) formed in a particular size by filling amodified cross-section hollow fiber with cells of interest exertscellular functions efficiently without causing a necrotic layer in thefilled cells.

That is, for achieving the above objects, the inventors of the presentinvention have advanced their research and development furthermore and,as a result, they have given thought to the facts that: (1) the innerdiameter of the hollow fiber membrane is not restricted as far as amodified cross-section hollow fiber being deformed is used instead ofthe perfect circle-shaped hollow fiber membrane commonly used in theart, while a distance between the cells and the inner wall of the hollowfiber membrane is kept at a far enough distance to prevent the cellsfrom causing necrosis; and (2) the commercially available hollow fibermembrane, which has been known to be made of many different materialsand to have permeating properties, can be used when the modified crosssections as mentioned above is made by deforming the perfectcircle-shaped hollow fiber membrane. Furthermore, the inventors of thepresent invention have given thought to the facts that the affinitybetween the cells and the membrane and the substance permeability of thecell-filled device of a modified cross-section hollow fiber membranetype can be designed without restriction, so the device can find use ina remarkably increased number of applications including implantable andcirculation type hybrid artificial organs. No reports have beenconventionally made on cell aggregates (organoids) each using such amodified cross section hollow fiber.

The present invention pertains to the following (1) to (29).

(1) A cell-filled device of a modified cross-section hollow fibermembrane type, including hollow fiber membranes whose hollow portionsare filled with cells, characterized in that:

the hollow fiber membranes have modified cross sections; and

a cell aggregate provided in each of the hollow portions has cellsformed into two or more layers in arbitrary directions, provided that adistance from an arbitrary point of the cell aggregate to the nearestinner wall of the hollow fiber membrane is less than 75 μm.

(2) The cell-filled device of a modified cross-section hollow fibermembrane type according to (1), in which the distance to the nearestinner wall of the hollow fiber membrane is 50 μm or less.

(3) The cell-filled device of a modified cross-section hollow fibermembrane type according to (1) or (2), characterized in that across-section of the modified cross-section hollow fiber membrane is ina flat form.

(4) The cell-filled device of a modified cross-section hollow fibermembrane type according to any one of (1) to (3), characterized in thata pore size of the hollow fiber membrane is 0.001 to 5 μm.

(5) The cell-filled device of a modified cross-section hollow fibermembrane type according to (4), characterized in that the pore size is0.05 to 1 μm.

(6) The cell-filled device of a modified cross-section hollow fibermembrane type according to any one of (1) to (5), characterized in thatthe hollow fiber membrane is made of a synthetic polymer having acontact angle of 70 degrees or less.

(7) The cell-filled device of a modified cross-section hollow fibermembrane type according to (6), in which the synthetic polymer is athermoplastic resin.

(8) The cell-filled device of a modified cross-section hollow fibermembrane type according to (7), in which the thermoplastic resin is apolyethylene-based resin.

(9) The cell-filled device of a modified cross-section hollow fibermembrane type according to any one of (1) to (8), characterized in thatat least an inner surface of the hollow fiber membrane contains ahydrophilic polymer.

(10) The cell-filled device of a modified cross-section hollow fibermembrane type according to any one of (1) to (9), characterized in thatthe cells are cells derived from an animal tissue.

(11) The cell-filled device of a modified cross-section hollow fibermembrane type according to (10), characterized in that the cells derivedfrom an animal tissue are at least one kind of cell selected from thegroup consisting of cells derived from a liver, cells derived from aspleen, stem and precursor cells thereof, and genetic recombinant cells.

(12) The cell-filled device of a modified cross-section hollow fibermembrane type according to (11), characterized in that the cells derivedfrom an animal tissue are hepatic cells.

(13) The cell-filled device of a modified cross-section hollow fibermembrane type according to any one of (10) to (12), characterized inthat the cells derived from an animal tissue are cells derived from ahuman organ.

(14) A cell-filled device, including hollow fiber membranes and cells,the device being provided as the cell-filled device of a modifiedcross-section hollow fiber membrane type for implantation according toany one of (1) to (13), in which each of the hollow portions contains acell aggregate and both ends of each hollow fiber membrane are sealed.

(15) A cell-filled device of a modified cross-section hollow fibermembrane type for a hybrid artificial organ, which is one according toany one of (1) to (13).

(16) A hybrid artificial organ, including at least one cell-filleddevice of a modified cross-section hollow fiber membrane type accordingto any one of (1) to (13).

(17) A hybrid artificial organ, including at least one cell-filleddevice of a modified cross-section hollow fiber membrane type accordingto any one of (1) to (13), being housed in a container having an inletand an outlet for a liquid to be treated, characterized in that aninside of a hollow of the cell-filled device of a modified cross-sectionhollow fiber membrane type is separated from an external of the hollowforming a communication path of the liquid to be treated.

(18) A method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type, including the steps of:

a) obtaining a modified cross-section hollow fiber membrane; and

b) injecting a cell suspension into a hollow of the hollow fibermembrane.

(19) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to (18), furtherincluding the step of producing the hollow fiber membrane using doubleannular spinning nozzle having a modified cross section.

(20) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to (18), furtherincluding the step of applying an external force for deformation to ahollow fiber membrane not having a shape of interest in an approximatelyvertical direction of its fiber axis to obtain the modifiedcross-section hollow fiber membrane.

(21) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to (18), furtherincluding the step of drafting the hollow fiber membrane not having ashape of interest in the direction of its fiber axis while deforming theshape of the cross section to mold the membrane into the modifiedcross-section hollow fiber membrane.

(22) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to any one of (18) to(21), characterized in that the cross section of the modifiedcross-section hollow fiber membrane is in a flat form.

(23) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to any one of (18) to(22), characterized in that a material of the hollow fiber membrane is athermoplastic resin.

(24) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to any one of (18) to(23), characterized in that injected cells are cells derived from ananimal tissue.

(25) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to (24),characterized in that the cells derived from an animal tissue are atleast one kind of cell selected from the group consisting of cellsderived from a liver, cells derived from a spleen, stem and precursorcells thereof, and genetic recombinant cells.

(26) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to (25),characterized in that the cells derived from an animal tissue arehepatic cells.

(27) The method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to any one of (24) to(26), characterized in that the cells derived from an animal tissue arecells derived from a human organ.

(28) A method of manufacturing a hybrid artificial organ including themethod of manufacturing a cell-filled device according to any one of(18) to (27).

(29) A method of manufacturing a hybrid artificial organ characterizedin that:

at least one modified cross-section hollow fiber membrane used in themethod of manufacturing a cell-filled device according to any one of(18) to (27) is housed in a container having an inlet and an outlet fora liquid to be treated, and an injection opening for cells; and

potting is performed such that an inside of a hollow is communicatedwith the injection opening for cells and separated from an externalportion of the hollow, followed by injecting cells into hollow portionsto form a cell aggregate.

According to the present invention, the device can function efficientlyas a hybrid artificial organ because cells filled in the modifiedcross-section hollow fiber function efficiently without waste andwithout causing any necrotized layer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the cross section of a modifiedcross-section hollow fiber membrane of the present invention.

FIG. 2 is a schematic diagram showing a manufacturing method of themodified cross-section hollow fiber membrane of the present invention.

FIG. 3 is a photomicrograph showing the cross section of the modifiedcross-section hollow fiber membrane of the present invention.

FIG. 4 is an electron photomicrograph showing the surface of a PE/EVALmodified cross-section hollow fiber membrane of the present invention.

FIG. 5 is an electron photomicrograph showing the surface of a PPmodified cross-section hollow fiber membrane of the present invention.

FIG. 6 is a photomicrograph showing the cross section of a cell-filleddevice of a modified cross-section hollow fiber membrane type of thepresent invention (on the 3rd day of culture, HE staining).

FIG. 7 is a graphical representation showing the time curve of cellmaintenance ratio due to the cell-filled device of a modifiedcross-section hollow fiber membrane type of the present invention.

FIG. 8 is a graphical representation showing a rate of ammonia removaldue to the cell-filled device of a modified cross-section hollow fibermembrane type of the present invention.

FIG. 9 is a graphical representation showing a rate of albumin secretiondue to the cell-filled device of a modified cross-section hollow fibermembrane type of the present invention.

FIG. 10 is a photomicrograph showing the effects of imparting ahydrophilic nature to the surface on the formation of a cell aggregate(organoid) in the modified cross-section hollow fiber membrane of thepresent invention(on 1 hour of incubation).

FIG. 11 is a photomicrograph showing the effects of imparting ahydrophilic nature to the surface on the cellular attachment in themodified cross-section hollow fiber membrane of the present invention(on the 1st day of incubation).

FIG. 12 is a schematic diagram showing a hybrid artificial organ of thepresent invention.

FIG. 13 is a schematic diagram showing a state where cells are filledand immobilized in a modified cross-section hollow fiber membraneinstalled in the hybrid artificial organ of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

The modified cross-section hollow fiber membrane of the presentinvention is not intended for a cross-sectional structure generally usedfor a blood purifying membrane, a ultra filtration membrane, or the likeand having inner and outer peripheral portions which are in the shapesof perfect circles and arranged concentrically. In this case, however,the cross-sectional structure of at least an inside of the hollow fibermembrane is intentionally deformed particularly in order to define adistance from the arbitrary point of the cell aggregate (organoid)formed in the hollow portion to the nearest inner wall of a hollow fibermembrane within a specific range. As shown in FIG. 1, specific examplesof the shape include a flat shape, an oval shape, polygonal shapes suchas triangle, quadrangle, and pentagonal forms, and infinite forms suchas comma-shaped bead and star forms. Of those, in consideration of theeasiness of filling with the cells and the handling of the membrane, theflat or oval shape is preferable. The flat shape is particularlypreferable.

The modified cross-section hollow fiber membrane is preferably designedsuch that the forms of inner and outer peripheral portions are almostequal and concentric with each other (in other words, the membranethickness thereof is almost uniform). For any purpose, the inner andouter peripheral portions may have different forms.

The term “modified cross section” used herein represents the shape of across section obtained by cutting the hollow fiber membrane in the axialdirection of the fiber. For instance, the shape of a cross sectionmodified in the axial direction of the fiber, which is described in theprior art (JP 63-233777 A) is also out of category. This is because sucha shape does not attain the object of keeping a space between the cellsand the inner wall of the hollow fiber membrane of the present inventionat a distance far enough to prevent the cells from causing necrosisthereof, is hardly formed uniformly, and hardly permits the filling ofcells.

As a structure of a membrane portion, the modified cross-section hollowfiber membrane may use any conventionally known hollow fiber membranestructures such as a sponge structure, a uniform structure, and amacrovoid structure. In addition, the structure of the modified hollowfiber may be straightened out in the axial direction of the fiber, orcrimped in waves.

Preferably, the material of the modified cross-section hollow fibermembrane used in the present invention is a thermoplastic resin from theviewpoint of the deforming process described later. Examples of thematerial include polyolefin-based, polyester-based, polysulfone-based,polyethersulfone-based, polypropylene-based, polyethylene-based,polyacrylonitrile-based, polymethylmethacrylate-based, polyvinylchloride-based, and polyamide-based resins. The reason of describingthose materials as “ - - - based” is that the polymer mentioned abovemay be provided as a main component and, for any purpose, a secondarycomponent may be blended or introduced by graft polymerization.Alternatively, the secondary component may be any copolymer includingrandom and block copolymers.

The modified cross-section hollow fiber membrane used in the presentinvention is made of the thermoplastic resin described above, preferablya synthetic polymer having a contact angle of 70 degrees or less. Here,the term “contact angle” of the synthetic polymer is defined as follows.That is, a synthetic polymer is uniformly applied on a support in ahorizontal position such as a uniform film made of a synthetic polymeror a glass plate, and then dried to obtain a product. Then, pure wateris dropped on the product to form a liquid droplet. At this time, thecontact angle is an angle on the liquid-including side among angles thata flat surface makes with a tangent at a contact point among threephases, liquid droplet, synthetic polymer surface, and gas phases, whichis a tangent to the liquid.

Making the hollow fiber membrane of such a synthetic polymer ispreferable because, in particular, the cells can be prevented fromattaching on the inner surface at some degree and a decrease insubstance permeability of the membrane due to the cellular attachmentcan be reduced. In addition, it is also preferable in terms of asubstance exchange through the membrane because of an improvement inwettability of the hollow fiber membrane with the liquid to be treated,such as blood, plasma, or a physiological solution.

The synthetic polymer that satisfies a contact angle of 70 degrees orless can be suitably selected from the thermoplastic resins describedabove or may be one obtained by applying the thermoplastic resindescribed above on a membrane made of any material.

In the present invention, at least an inner surface of each of thosemodified cross-section hollow fiber membranes, or a contact surface withat least cells may contain a hydrophilic polymer for the purpose ofcontrolling the affinity for cells.

Examples of the hydrophilic polymer include: hydrophilic syntheticpolymers such as polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, ethylene-vinyl alcohol copolymer, polyethylene imine, andpolyallyl amine; and hydrophilic polysaccharides such as cellulose,chitosan, agarose, dextran, and dextran sulfate. However, the polymer isnot limited to those polymers. Any polymer may be used as long as it iscapable of controlling the viability of the cells being attached.

Of those, exceptionally preferable is the ethylene-vinyl alcoholcopolymer. According to the findings of the inventors of the presentinvention, the ethylene-vinyl alcohol copolymer shows a high rate of theorganoid formation of hepatic cells at an early stage, while showing alow enough cellular attachment property to prevent the loss of substancepermeability of the membrane.

Any of those hydrophilic polymers may be introduced into at least aninner surface of the modified cross-section hollow fiber membrane bymeans of any of blending, graft polymerization, copolymerization, or thelike as well as coating. Of course, it may be introduced into the entiremembrane.

For the substance permeability of the modified cross-section hollowfiber membrane, a pore size may be as large as possible in terms of amass transfer. In consideration of the leakage of cells, the pore sizemay be small. For satisfying both points, the pore size is in the rangeof 0.001 to 5 μm, which can be suitably selected on the basis of themolecular weight of the material and the size of cells for the purposeof detoxification or removal. In consideration of the supply of aculture solution containing various nutritive substances, i.e.,permeability of water and nutrients, the pore size is preferably in therange of 0.05 to 1 μm.

The cell-filled device of a hollow fiber membrane type of the presentinvention is obtained by: filling the inside of the hollow portion ofthe modified cross-section hollow fiber membrane with cells; and thenaggregating the cells to provide a cell aggregate (organoid). Forretaining the cells to be utilized in the hollow portion, there is aneed for a means of injecting cells. However, the form and mechanismthereof are not particularly limited, as will be described in amanufacturing method later. Thus, those matters may be appropriatelychosen and used.

The term “cell aggregate” (organoid) as used herein is a multicellularaggregate made of cells being accumulated, which approximates an organhaving functions originally found in the cellular tissue. For instance,a multicellular aggregate made of hepatic cells being accumulated, whichare obtained in the present invention, can be referred to as a cellaggregate (organoid) because it exhibits hepatic functions such as anammonia removal effect and an albumin secretion effect, which areinherent functions of the liver tissue. The cell aggregate (organoid) asused herein is not limited to a cell aggregate formed by filling anddensifying the cells by means of a dynamical procedure such ascentrifugal forces or pressure as defined in JP 2002-182677 A. Forinstance, as disclosed in JP 10-33671 A, there is also a cell aggregateformed by: filling hepatic cells in an inner cavity of a hollow fiber;incubating the resultant in a perfusion solution; growing the hepaticcells in the inner cavity of the hollow fiber to produce anextracellular matrix; and then proceeding cell-cell adhesion andintercellular matrix-cell adhesion.

The cells used in the present invention are cells derived from thetissue of an animal. Depending on the collection portion, examples ofthe cells include: organ-derived cells such as cells derived from theliver (containing at least one of hepatic cells, endothelial cells,Kupffer cells, and fibroblast cells), cells derived from the spleen,heart muscle cells, cells derived from the kidneys; and tissue cellssuch as skin cells, epidermal keratinocytes, fibroblast cells, vascularendothelial cells, vascular wall cells, nerve cells, and cartilagecells. Those cells may be combined with each other to use. In addition,depending on the development stage, in addition to those mature cells,stem cells or precursor cells may be used. Furthermore, for the normalcells, genetic recombinant cells may be used. Examples of geneticrecombinant cells include cells being immortalized by the introductionof immortalizing genes such as Tert, Bmi1, Large T of SV40, and Bc12.

Of those, the cells used in the cell-filled device of a modifiedcross-section hollow fiber membrane type of the present invention aremost preferably hepatic cells. The liver is one of the larger organs.The liver has a wide variety of complicated functions. For example, theliver relates to by the synthesis, or storage of materials required forthe living body, such as proteins and sugars, or metabolic detoxicationfor ammonia, drugs, or the like, and the digestion of fat and theabsorption of vitamins by the release as an external secretory organ ofbile acid or the like. Therefore, the device using hepatic cells can bevery useful in terms of functions in spite of being small.

The cell sources of the present invention include normal hepatic cellsderived from laboratory mice, rats, guinea pigs, rabbits, dogs, pigs,baboons, and humans but not limited to these sources. The establishedhepatic cells may be also provided as targets.

In the case of normal hepatic cells, isolated hepatic cells can beobtained using a general enzymatic digestion method by which the liveris treated with an enzyme solution such as a collagenase solution.

The cell density filled in the modified cross-section hollow fibermembrane is preferably 1×10⁷ cells/cm³ or more. When the cell density isequal to or more than that, the device can be compacted when it is usedas the implantable or circulation type hybrid artificial organ.

By the way, when the cells are hepatic cells in particular, the celldensity is preferably more than 5×10⁷ cells/cm³. The cell density in theliving human liver is 1 to 2×10⁸ cells/cm³. Thus, it is desired that thecell density of the hepatic cell aggregate (organoid) be close to thecell density of the living liver. In addition, when the cells are filledat high density by means of centrifugal forces and hydrostaticpressures, the filling can be attained as the load thereof is high.However, when the load is too high, the hepatic cells themselves willreceive damage or die, so the functions of the hepatic cells cannot bekept. Therefore, in the case of the hepatic cell aggregate (organoid),the density of the cells is more than 5×10⁷ cells/cm³ but not more than2×10⁸ cells/cm³, preferably 8×10⁷ cells/cm³ or more, more preferably9×10⁷ cells/cm³ or more.

The cell aggregate (organoid) of the present invention should becomposed of two or more layers of cells being accumulated in arbitrarydirections. As long as the cell aggregate has a thickness correspondingto the two or more layers, the functions of the cell aggregate(organoid) can be exerted. The term “two or more layers of cells beingaccumulated in arbitrary directions” as used herein means that the cellsare accumulated to form two or more layers even though the cellaggregate (organoid) is cut any radial direction. The term “a distancefrom an arbitrary point of the cell aggregate to the nearest inner wallof the hollow fiber membrane is less than 75 μm” means that a distancefrom any point of the cell aggregate (organoid) formed in the hollowportion to the inner wall of the hollow portion cannot be 75 μm or more.If it becomes 75 μm or more, the supply of oxygen cannot reach the cellslocated in the center of the cell aggregate(organoid), thereby causingnecrosis. Therefore, the thickness of the cell aggregate (organoid) inthe present invention should correspond to two or more layers. Inaddition, a distance from any point of the cell aggregate (organoid)formed in the hollow portion to the inner wall of the nearest hollowfiber should be less than 75 μm. Here, the term “thickness” means athickness from the surface layer of the cell aggregate (organoid) to theopposite of the surface of the cell aggregate. A thickness (length) ofthe cell aggregate (organoid) formed in the modified hollow fibermembrane can be appropriately set in the axis direction of its fiber. Inaddition, when the modified hollow fiber membrane is in a flat form, forexample, the thickness in the major axis direction is defined at will asfar as the thickness in the minor axis direction satisfies the abovedescription.

When the cell aggregate (organoid) has a higher cell density, the gapsbetween cell to cell decreases. Thus, the thickness cannot be increasedsubstantially because of the need for supplying oxygen. On the otherhand, even though the cell density is within the range of the presentinvention, a larger thickness is preferable when the cell density iscomparatively small. Therefore, in the cell aggregate (organoid) of thepresent invention, it is more preferable to establish a reversedcorrelation between the cell density and the thickness.

In the cell aggregate (organoid) of the present invention, except thesurface thereof, it is important that the respective cells contact eachother in a three-dimensional perspective manner. In the living liver, ithas been recognized that hepatic cells express the functions thereof byexchanging information between the adjacent cells through variouscell-cell connections. In the present invention, the hepatic cells aresubjected to physical forces such as centrifugal forces and hydrostaticpressures when filled at high density. Therefore, a tissue-like bodyhaving a high cell density can be formed by considerably improving thefrequency of contact between the cells.

It has been also possible to form a cell aggregate by filling the cellsin the inner cavity of the hollow fiber and then growing the cells tomake the cells be in close contact with each other.

In addition, the cell aggregate (organoid) of the present invention ispreferably provided with a skin layer on the surface of the accumulatedbody thereof. When the hepatic cells are subjected to physical forcessuch as centrifugal forces and hydrostatic pressures, and then incubatedfor 3 to 5 days, the cells on the surface layer of the cell aggregate(organoid) become flattened and the surface layer of the cell aggregate(organoid) becomes smooth, causing the emergence of a skin layer. Theskin layer may be caused by the cellular state of the surface layer ofthe cell aggregate (organoid) and a cellular secretion product.

Next, a hybrid artificial organ using the cell-filled device of amodified cross-section hollow fiber membrane type will be described.

In a module obtained such that at least one of the modifiedcross-section hollow fiber membranes of the present invention is housedin a container having an inlet and an outlet for a culture medium or aliquid to be treated, and an injection opening for cells, and inaddition, the inside of the hollow is communicated with the injectionopening for cells, and furthermore, the inside of the hollow is pottedso as to be separated from the external portion of the hollow that formsa passage of the liquid to be treated. Therefore, a cell aggregate(organoid) formed by filling the cells to be utilized into the hollowportion of the hollow fiber membrane will be used as a hybrid artificialorgan. FIG. 12 and FIG. 13 show schematic diagrams of a hybridartificial organ of the present invention, in which a modifiedcross-section hollow fiber is incorporated.

As shown in FIG. 12, a container 2 having an inlet and an outlet (5 and6) for a culture solution or a liquid to be treated has at least onemodified cross-section hollow fiber membrane 1 being filled. Both endsof the modified cross-section hollow fiber membrane are subjected to apotting process and fixed such that the inside of the hollow iscommunicated with the injection opening for cells in sealing portions 3,while being separated from the external portion of the hollow forming apassage for the liquid to be treated.

For the container and potting structure, any of container materials andshapes generally used for hollow fiber membrane type blood purifiers isavailable. For instance, a suitable container material is one havinghigh strength and high transparency and provides excellent safety, whichis typified by a polycarbonate-based resin or a polystyrene-based resin.Additionally, but not particularly limited to, a cheap polyolefin-basedresin or any of various copolymer resins may be used. In addition, thecontainer may be shaped such that a body portion is cylindrical and aflow channel of the liquid to be treated is provided in the vicinity ofboth ends thereof. In the container, at least one cell-filled device ofa modified cross-section hollow fiber membrane type of the presentinvention is installed.

Furthermore, the potting structure is not particularly limited, and mayhave any of configurations. For instance, like a hollow fiber membranetype blood purifier, the potting structure may be one in which both endsof the hollow fiber membrane in the container are potted and then theinner and outer sides of the membrane are separated from each other.Alternatively, like an example of an endotoxin cut filter, both endsthereof are potted or one of the ends is provided as a sealed end.Furthermore, alternatively, like an example of a house-hold waterpurifier of a hollow fiber membrane type, the hollow fiber membrane isinstalled in the container in the shape of letter U and one end thereofis potted.

The above module is provided with at least one injection opening 4 forcells to introduce cells into the module in addition to the inlet andoutlet (5 and 6) for a culture solution or a liquid to be treated. Thus,a cellular suspension is introduced from the injection opening 4 forcells and then introduced into the hollow portion of each of the hollowfiber membranes through an opening end of the hollow fiber membrane atthe sealing portion 3. Another sealing portion 3 on the opposite end maybe provided such that the opening end of the hollow fiber membrane issealed in advance or sealed after the introduction of cells. Asdescribed above, after the cells are introduced into the hollow portionof the hollow fiber opening end, the cells can be immobilized in thehollow portion by sealing the sealing portion 3 on the cell injectionside with a treatment of sealing the injection opening 4 for cells, orthe like. The resultant is a so-called hybrid artificial organ in thepresent invention. The appearance of cells immobilized in the hollowportion is schematically shown in FIG. 13.

In this module, in the container 2, a culture solution flows in a spaceformed between the external side of the hollow fiber membrane and thesealing portion through the culture medium inlet 5 and the culturemedium outlet 6. Here, the culture solution, which is filled in theinside of the modified cross-section, is used for supplying oxygen andnutrients to cells forming the cell aggregate (organoid) and for theremoval of a metabolic decomposition product. In this space,furthermore, blood, plasma, a diluent thereof, or a blood preparation,provided as a liquid to be treated, may flow.

The hybrid artificial organ having a container structure as describedabove has a modified cross-section hollow fiber membrane as an installedhollow fiber membrane. Thus, the liquid to be treated tends to uniformlyflow in the space between the membranes. In addition, it is excellent inthe efficiency of substance exchanges such as detoxification andremoval. Besides, it is suitable for a hybrid artificial organ capableof maintaining the functions for a long time. In addition, the hybridartificial organ can be also obtained by appropriately deforming ahollow fiber membrane commercially available. Consequently, there is anadvantage of utilizing cell aggregates (organoid) filled in membranesmade of various materials and having substance permeability.

Next, a method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type of the present invention willbe described.

For manufacturing the cell-filled device of a modified cross-sectionhollow fiber membrane type of the present invention, at first, a step ofobtaining a modified cross-section hollow fiber membrane is needed.

The modified cross-section hollow fiber membrane of the presentinvention can be prepared using any of the fiber-spinning methods knownin the art, such as a wet process, drying process, and a meltingprocess. However, a method may be appropriately selected so as to besuitable for the membrane materials used.

The methods of obtaining the modified cross-section hollow fibermembrane include a method for the formation of a modified cross sectionsimultaneously with the membrane formation and a method for deforming ahollow fiber membrane by subjecting the hollow fiber membrane to amechanical processing after membrane formation. In the former, aspinning solution may be discharged from a double annular spinningnozzle having a modified cross section and then coagulated. The formeris preferable because modified cross-section hollow fiber membranes ofvarious shapes can be obtained by using a spinning nozzle having a crosssectional shape of interest. In addition, in the latter, a hollow fibermembrane that has already been formed may be subjected to a deformingforce from the substantially vertical direction of the fiber axis. Forexample, as shown in FIG. 2, the hollow fiber membrane can be cut intoappropriate lengths and deformed using a pressing machine having aspecific shape. Alternatively, a slit roller having a constant width anda specific form designed to sandwich a hollow fiber membrane ispreferably used because the hollow fiber membrane can be continuouslydeformed during the fiber-forming step or after the membrane-forming.Such a deforming process after the membrane formation can utilize anycommercially available hollow fiber membranes. Thus, even though theinner diameter of the hollow fiber member is so large that it isinherently unstable to the organoid, it can be deformed into desireddimensions by means of flattening or the like. Besides, it is verypreferable in that the advantages of membrane materials and substancepermeability can be used effectively. In any case, when an externaldeforming force is applied, a thermal setting may be preferably appliedat temperatures which do not deform the membrane material.

By the way, in particular, a melt spinning method using apolyolefin-based resin or a polyethylene-based resin may deform(flatten) the shape of the cross section simultaneously by drafting thehollow fiber membrane in the axial direction of the fiber using adrawing roller having a hardened surface in a drawing treatment which isgenerally carried out in the art. This is because the rate ofsolidification of the spinning solution is slow in melt spinning incomparison with wet spinning, so the membrane during the membraneformation can be easily deformed. Such a method is particularlypreferable because a specific spinning nozzle and post processing arenot needed.

Next, there is a need for a step of injecting a cell suspension into thehollow portion of the modified cross-section hollow fiber membrane. Inthis step, it is desired to provide means for injecting cells forintensed use into the hollow portion of the hollow fiber membrane inadvance. For instance, at least one end of the hollow fiber membrane issubjected to a resin potting process and then hardened, followed bycutting the resin so as to form an open end of the hollow fibermembrane. After the other end is sealed, the opening end may be providedwith a fixture, nozzle, or the like for injecting a cell suspension intothe opening end. However, the sealing means, injection fixture, and soon are not particularly limited.

For obtaining the hybrid artificial organ by filling and immobilizingcells after housing the modified cross-section hollow fiber membrane inthe container, the resulting modified cross-section hollow fibermembrane is installed in a cylindrical container having an inlet and anoutlet for a culture solution or a liquid to be treated and then atleast one end thereof is potted. As exemplified previously, the pottingform is not particularly limited. Potting may be performed on both endsor may be performed on one end mounted with a U-shaped hollow fibermembrane, which may be separated so as to prevent a short pass insideand outside the hollow membrane members. Next, after the potting resinis hardened, the end of the resin is cut to form an opening end of themodified cross-section hollow fiber, followed by mounting a header caphaving an injection opening for cells. A series of molding methods maybe carried out on the basis of a method of molding a well known bloodpurifier, water purifier, or the like of a hollow fiber membrane type,and is not particularly limited. In this case, before the injection ofcells, an opening end or a header nozzle opposite to the injectionopening for cells is sealed to prevent the injected cells from flowingout.

Subsequently, when the cell suspension is injected into the hollowportion of the hollow fiber membrane, cells being dispersed aresubjected to external forces such as centrifugal forces and hydrostaticpressures to allow a liquid element to be removed through filtrationwhile filling a high cell density for incubation. Concretely, the cellsare placed in the hollow fiber and then packed at high density by aneffect of physical forces such as centrifugal forces and hydrostaticpressures. In the case of a method of loading with centrifugal forces,the cells are accumulated at high density and then incubated for apredetermined time to obtain the cell aggregate (organoid). Similarly,in the case of a method of loading with hydrostatic pressures, the cellsare loaded with the hydrostatic pressures and incubated to obtain thecell aggregate (organoid). Alternatively, hepatic cells are filled inthe inner cavity of the hollow fiber to grow hepatic cells by means of aperfusion and to produce an intracellular matrix. Furthermore, thecell-cell adhesion or the cell-intercellular matrix adhesion can beproceeded to obtain a cell aggregate.

It is preferable to prepare a cell suspension before injection into thehollow fiber in order to prevent damage to the cells. For attaining ahigh density without damaging the cells, the concentration of the cellsuspension is preferably 2×10⁷ cells/ml or less, more preferably 0.1 to1×10⁷ cells/ml. For filling at high density, it is preferable to performthe injection while removing only the culture solution from a pore ofthe hollow fiber membrane.

After the cells are injected into the hollow portion of the hollow fibermembrane, in substantially the axial direction of the hollow fibermembrane, a centrifugal force of 5 to 1,500 G is applied for about 30 to600 seconds to accumulate the cells at high density. If the centrifugalforce exceeds 1,500 G, the cells will be damaged or killed. Forpreventing the cells from being killed, a suitable centrifugal force maycorrespond to a load of about 60 G×90 seconds.

In the case of using hydrostatic pressure, the hollow fiber injectedwith cells is set up and then a hydrostatic pressure of 5 to 25 kPa isloaded in the hollow fiber for 4 to 120 hours. Most preferable is toload with a constant hydrostatic pressure of 10 kPa for 24 hours.

The cells are filled as described above and then the cell aggregate(organoid) is formed by incubating the cells, resulting in a cell-filleddevice of the present invention.

The culture solution used may be a serum-free medium prepared by addinghormones and inorganic salts to a basic medium such as William E Medium(WE) or Dulbecco's Modified Eagle Medium (DMEM) or a serum-containingmedium prepared by adding serum to the basic culture of WE, DMEM, or thelike.

As described above, in the present invention, the immobilizedcell-filled device having cells filled in the modified cross-sectionhollow fiber membrane is effectively used as a hybrid artificial organbeing modularized and installed in the container.

Furthermore, for instance, a hollow fiber membrane portion is collectedfrom the modified cross-section hollow fiber membrane or the hybridartificial organ after the formation of a cell aggregate (organoid) andthen both ends thereof are sealed with a procedure so as to preventcells from leaking. The resultant can also be suitably used.

The sealing form of the hollow fiber membrane is not particularlylimited as long as the cells being filled are not leaked from theopening portion of the membrane. For instance, several-millimeter toseveral-centimeter parts of both ends of the hollow fiber membrane maybe pressed, bent, thermally welded, embedded with a potting material, orthe like. In particular, it is more preferable that the hollow fibermembrane be made a biodegradable polymer known in the art because it canbe used as an implant use type device.

The cell-filled device of a modified cross-section hollow fiber membranetype having such a sealing structure and composed of the hollow fibermembrane and cells is also favorably used as a device for an artificialorgan capable of maintaining its functions for a long time. In addition,the device can be attained by suitably deforming a commerciallyavailable hollow fiber membrane. Thus, there is an advantage in that thecell aggregate (organoid) filled in the membrane made of any of variousmaterials and having substance permeability can be used.

EXAMPLES

Hereinafter, the present invention is more specifically described withreference to examples, but the present invention is not limited to them.

The preparation of hepatic cells used for forming a cell aggregate(organoid) in the present invention as well as the measurement of thefunctional activity of the resulting hepatic cell aggregate (organoid)were performed as follows.

(Preparation of Hepatic Cells)

For preparing primary rat hepatic cells, 150 ml of a solution of 0.5mg/ml collagenase (manufactured by Wako Pure Chemical Industries, Ltd.)was prepared. After a cannula was introduced into the portal vein (theblood vessel which leads to the liver) of a male Wistar-line rat at 7weeks of age (weight: 250 g) and the blood was drawn for 5 minutes at 30ml/min, the collagenase solution heated to 37° C. was fed thereinto for10 minutes at 15 ml/min. The liver treated with collagenase was put intoa culture solution and the hepatic cells were dispersed using a scalpeland a pipette. The resulting hepatic cell suspension was washed threetimes to remove cells with the exception of hepatic cells (at a puritygreater than 95%). The hepatic cell suspension having the final densityof 2.0×10⁶ cells/ml was made and used for a culture experiment.

(Rate of Ammonia Removal)

Ammonia was added to a culture medium to bring the concentration to 1 mMand temporal amounts of the reduced ammonia concentration were measuredto calculate a rate of ammonia removal (μmol/10⁶ immobilized cells/day).

(Rate of Albumin Secretion)

Albumin secreted into the culture medium was quantified by an enzymelabeled immunoassay and converted into a rate of albumin secretion pernumber of initial immobilized unit cells (μg/10⁶ immobilized cells/day).

Example 1

Using a polyethylene hollow fiber membrane coated with an ethylene vinylalcohol copolymer (hereinafter, referred as to a PE/EVAL hollow fiber,330 μm in inner diameter, 50 μm in membrane thickness) and apolypropylene hollow fiber membrane (hereinafter, referred as to a PPhollow fiber, 330 μm in inner diameter, 50 μm in membrane thickness),modified cross-section hollow fiber membranes were made by a flatplate-pressing manner shown in FIG. 2 (1). In application of a heat setby sandwiching the above hollow fiber membranes and stainless spacers,each having a thickness of 100 μm between two glass plates and leavingthem to stand at 120° C. for 6 hours with the central portion of theglass plates fixed with clips, the modified cross-section hollow fibermembranes were created. The modified cross-section hollow fibermembranes that were made were denoted as PE/EVAL hollow fiber-press 100and PP hollow fiber-press 100, respectively.

After the resulting modified cross-section hollow fiber membranes wereembedded into liquid silicon, thin sections were made, thereby observingthe cross-sectional shape. Moreover, using a scanning electronmicroscope (SEM), the surface structure of the resulting modifiedcross-section hollow fiber membranes was observed. The observations areshown in FIGS. 3 to 5.

Next, for the functional evaluation of a hepatic cell aggregate(organoid), made were modified cross-section hollow fiber membranebundles of a bundle composed of nine PE/EVAL-press 100 hollow fiberseach having a length of 5 cm and a bundle composed of six PP-press 100hollow fibers each having a length of 5 cm. One end of each bundle wasattached with a port for injecting cells and the other end thereof washermetically sealed.

First, 0.6 ml of the cell suspension at 2.0×10⁶ cells/ml was injectedinto those modified cross-section hollow fiber membrane bundles througha cell injection opening using a syringe, simultaneously by fillingcells into the modified cross-section hollow fiber membranes whileremoving the culture solution from pores of the hollow fiber membranesby filtration. Next, in order to decrease space among the cells,high-density filling was performed by centrifugation treatment at 60×Gfor 90 minutes to induce the formation of the hepatic cell aggregate(organoid).

After the completion of centrifugation, the hollow fiber membranes werecut out at the position of 3 cm from the bottom end of the bundles inwhich the cells was filled in a very dense state, and were accommodatedin a culture dish of 35 mm in diameter (manufactured by Falcon). To theculture dish, 2 ml of a serum-free medium of 13.5 g/L Dulbecco'smodified eagle medium (manufactured by GIBCO) supplemented with 60 mg/Lproline, 50 ng/ml EGF (manufactured by Funakoshi), 10 mg/Linsulin(manufactured by SIGMA), 7.5 mg/L hydrocortisone (manufactured by WakoPure Chemical Industries, Ltd.), 0.1 μM copper sulfate pentahydrate(manufactured by Wako Pure Chemical Industries, Ltd.), 3 μg/L selenicacid (manufactured by Wako Pure Chemical Industries, Ltd.), 50 pM zincsulfate heptahydrate (manufactured by Wako Pure Chemical Industries,Ltd.), 50 μg/L linoleic acid (manufactured by SIGMA), 58.8 mg/Lpenicillin (manufactured by Meiji Seika), 100 mg/L streptomycin(manufactured by Meiji Seika), 1.05 g/L sodium bicarbonate (manufacturedby Wako Pure Chemical. Industries, Ltd.), and 1.19 g/L HEPES(manufactured by Dojindo) was added and rotation culture was performedon a shaker at 45 rpm in 5% carbon dioxide and 95% air atmosphere.

For observing the culture state of the hepatic cells, the hepatic cellsfilled in the hollow fiber bundles were immobilized in a 10% neutralbuffered formal in solution in each period of culture. Subsequently, theimmobilized hepatic cells were paraffin-embedded and thin sections weremade, followed by observing the distribution of living cells and deadcells by hematoxylin-eosin staining. In addition, the hepatic cells werehomoginized with a polytron homogenizer together with hollow fiberbundles in each period of culture, and nuclei leaked out therefrom werestained with crystal violet to determine the change in the number ofcells by enumerating the number of nuclei. The culture state is shown inFIG. 6 and the change in the number of cells is shown in FIG. 7.

Furthermore, for the functional evaluation of the hepatic cells, ammoniawas added to a culture medium to bring the concentration to 1 mM andtemporal amounts of the reduced ammonia concentration were measured toevaluate their activity. Albumin secreted into the culture medium wasquantified to evaluate their activity. The results are shown in FIGS. 8and 9.

Comparative Example 1

Using a polyethylene hollow fiber membrane coated with an ethylene vinylalcohol copolymer (hereinafter, referred as to a PE/EVAL hollow fiberControl, 330 μm in inner diameter, 50 μm in membrane thickness) and apolypropylene hollow fiber membrane (hereinafter, referred as to a PPhollow fiber Control, 330 μm in inner diameter, 50 μm in membranethickness), a hepatic cell aggregate (organoid) was formed in the samemanner as in Example 1 except that the modification processing was notcarried out, to conduct the functional evaluation of hepatic cells.

Comparative Example 2

Using stainless spacers each having a thickness of 200 μm, modifiedcross-section hollow fiber membranes (denoted as PE/EVAL hollowfiber-press 200 and PP hollow fiber-press 200, respectively) were made.A hepatic cell aggregate (organoid) was formed in the same manner as inExample 1 except for a bundle composed of six PE/EVAL-press 200 hollowfibers each having a length of 5 cm, to conduct the functionalevaluation of hepatic cells. In this Comparative Example, a modifiedcross section having a minor axis of 150 μm in the inner peripheralportion of the membrane, i.e., a distance from an arbitrary point of thecross section to the inner wall beyond 75 μm was obtained.

The results from the foregoing Example 1 and Comparative Examples 1 and2 have demonstrated the following. As shown in FIG. 3 and Table 1, themodification pressing of the hollow fiber membrane produces a modifiedhollow fiber membrane having a minor axis of approximately 150 to 200 μmin the inner peripheral portion of the membrane by using a stainlessspacer of 200 μm in thickness and a modified hollow fiber membranehaving a minor axis of approximately 50 to 75 μm in the inner peripheralportion of the membrane by using a stainless spacer of 100 μm inthickness.

Moreover, as shown in FIGS. 4 and 5, in modification pressing in apressing manner, the modification pressing was found to be well madewithout causing the phenomena in which pores on the hollow fibersurface, even in the most deformable site, are crushed or completelytorn.

On the other hand, regarding the formation of a cell aggregate(organoid), as shown in FIG. 6, a cell aggregate (organoid) in whichhepatic cells were brought into close contact with each other was formedin any of the PE/EVAL hollow fiber-Control bundle, the PE/EVAL hollowfiber-press 200 bundle, and the PE/EVAL hollow fiber-press 100 bundle.However, the PE/EVAL hollow fiber-Control bundle (Comparative Example 1)and the PE/EVAL hollow fiber-press 200 bundle (Comparative Example 2),whose hollow fibers were perfect circle-shaped and had a larger minoraxis, were observed to have a dead cell layer (necrotized layer) in thecentral portion of the inside of the cell aggregate (organoid) likelydue to the depletion of oxygen. Accordingly, it is found that thefilling cells were all unable to be effectively utilized. In contrast,the PE/EVAL hollow fiber-press 100 bundle was not observed to have anecrotized layer in the central portion of the inside of the cellaggregate (organoid). As such, a minor axis of the hollow fiber isflattened to get smaller by modification pressing, and in thecross-sectional shape of a cell aggregate (organoid) formed in thehollow fiber, the distance from an arbitrary point of the cross sectionto the nearest inner wall of the hollow fiber is allowed to be less than75 μm. Consequently, even for a hollow fiber membrane originally havinga large inner diameter, a cell aggregate (organoid) which enables allcells to survive can be obtained by reducing an oxygen diffusion lengthin the cell aggregate (organoid).

For change in the number of cells, FIG. 7 shows a cell maintenance ratiowhich is regarded as the change in the number of cells in a hepatic cellaggregate (organoid) when the number of cells immediately afterinoculating is taken as 100%. This result indicates that the number ofcells can be satisfactorily maintained for a month or more in thePE/EVAL hollow fiber-press 100 bundle and the PP hollow fiber-press 100bundle in which a necrotized layer does not occur, as compared to thePE/EVAL hollow fiber-Control bundle in which a necrotized layer occursin the central portion.

Moreover, for the functional activity of the cell aggregate (organoid),as shown in FIGS. 8 and 9 regarding change in a rate of ammonia removaland a rate of albumin secretion per number of initial immobilized cells,the modified cross-section hollow fiber membrane bundle of the presentinvention maintains a better functional expression because no necrotizedlayer occurs in the hepatic cell aggregate (organoid).

From the above results, it is shown that the cell aggregate (organoid)using the modified cross-section hollow fiber membrane of the presentinvention enables the effective utilization of the filled cells withoutwaste.

Reference Example 1

As a hollow fiber membrane, a polyethylene hollow fiber membrane towhich a hydrophilic nature was imparted by coating its surface with anethylene vinyl alcohol copolymer (hereinafter, referred as to a PE/EVALhollow fiber, 330 μm in inner diameter, 50 μm in membrane thickness, 0.3μm in membrane pore size) was prepared. On the other hand, as a controlhollow fiber membrane, a hollow fiber membrane made of cellulosetriacetate having a surface more hydrophobic than the PE/EVAL hollowfiber (hereinafter, referred as to a CTA hollow fiber, 285 μm in innerdiameter, 50 μm in membrane thickness, 0.2 μm in membrane pore size) wasprepared. Using each hollow fiber, hollow fiber bundles composed of sixhollow fibers each having a length of 5 cm were made. One end of eachbundle was connected with a port for introducing cells and the other endthereof was hermetically sealed. The filling of cells into the hollowfiber membranes and culturing of them were performed in the same manneras in Example 1 except that 0.5 ml of a cell suspension at 4.0×10⁶cells/ml was injected.

The hepatic cells filled in the hollow fiber bundles were immobilized byleaving the bundles to stand in a 10% neutral buffered formalin solutionin each period of culture. Following the immobilization treatment, thehollow fibers were permeabilized to observe a cell form of the hepaticcell. Alternatively, the hollow fibers in which the hepatic cells werefilled were divided in the longitudinal direction using a scalpel andthe inner wall surface of the hollow fibers was exposed, followed byobservation using a low vacuum scanning electron microscope.

FIG. 10 shows the cell form of the hepatic cells in the PE/EVAL hollowfiber and the CTA hollow fiber at 1 hour of culture. Thus, in the CTAhollow fiber, hepatic cells were being filled in a very dense state, butthe formation of a cell aggregate (organoid) did not occur. On the otherhand, in the PE/EVAL hollow fiber to which a hydrophilic nature had beenimparted, a cell aggregate (organoid) was formed. Therefore, thedifference in the rate of cell aggregate (organoid) formation wasobserved.

Similarly, FIG. 11 shows the states of the inner wall surface of thePE/EVAL hollow fiber and the CTA hollow fiber on the first day ofculture, respectively. In the CTA hollow fiber, a cylindrical cellaggregate (organoid) was formed in the hollow fiber, although a numberof hepatic cells were observed to be attached to the inner wall of thehollow fiber. On the other hand, in the PE/EVAL hollow fiber, noattachment of cells to the inner wall of the hollow fiber was observedand all hepatic cells formed a cylindrical cell aggregate (organoid).

Upon reviewing the results, the treatment of imparting a hydrophilicnature to the surface of the hollow fiber membrane suppresses theattachment of cells to the hollow fiber membrane and is effective forthe early formation of a cell aggregate (organoid). In addition, thetreatment is effective because the reduction in membrane permeabilitycaused by the cellular attachment can be avoided.

The above results are summarized in Table 1. TABLE 1 Feature of modifiedhollow fiber created Mem- brane Minor axis [μm] Press thick- Pore BeforeAfter Hollow condi- ness size cell cell fiber Notation tion [μm] [μm]seeding seeding PE/EVAL Control None 50 0.3 330 hollow Press200 200-μm153 ± 22 214 ± 24 fiber spacer in use Press100 100-μm  48 ± 14 128 ± 22spacer in use PP Control None 50 0.5 330 hollow Press200 200-μm 200 ± 27235 ± 38 fiber spacer in use Press100 100-μm  74 ± 18 147 ± 29 spacer inuse

Example 2

A module having 167 elliptical modified cross-section hollow fibermembranes obtained in Example 1 (the above PP hollow fiber-press 100)filled in a housing container made of polycarbonate having a volume of1.79 cm³ was created. A schematic diagram of the created module is shownin FIG. 12. A modified cross-section hollow fiber membrane 1 was fixedwithin a container 2 using an urethane-based potting agent, while asealing portion 3 for completely blocking each space at the cellinjection side and at the medium perfusion side in the container 2 wasformed. Furthermore, a header cap provided with a cell injection opening4 was attached to the container 2. For performing cell seeding, thesealing portion 3 at the end of the module and the cell injectionopening 4 were completely sealed in advance.

Next, primary rat hepatic cells isolated by enzyme treatment wereprepared for a suspension at 4×10⁶ cells/ml. Thereafter, 13 ml of thecell suspension was injected into the hollow portion of the modifiedcross-section hollow fiber membrane through the cell injection opening4, followed by filling the cells into the hollow portion using thecentrifugation condition at 60×g for 90 seconds. After the cells werefilled, the cell injection opening 4 into which the cells were injected,was sealed, and the culture was performed by perfusing a culturesolution through a culture solution inlet 5 and a culture solutionoutlet 6.

After the completion of culture, the cross section of the modifiedcross-section hollow fiber membrane was observed in the same manner asin Example 1. As a result, it has been shown that almost all of the rathepatic cells filled in the hollow portions of the hollow fiber membranesurvived.

Thus, in the present invention, it has been indicated that a cellaggregate (organoid) can be obtained without generating a necrotizedlayer, even using a hybrid artificial organ installed with a cell-filleddevice of a modified cross-section hollow fiber membrane type.Additionally, it is suggested that such a module have effectiveness inuse for material production devices and cell incubators as well ashybrid artificial organs.

INDUSTRIAL APPLICABILITY

According to the present invention, it is shown that cells filled in ahollow fiber membrane efficiently work without waste by utilizing ahollow fiber membrane type cell-filled device. Furthermore, it issuggested that the device can be preferably utilized as an implantableor circulation type hybrid artificial organ accommodating them.

The hollow fiber membrane type cell-filled device of the presentinvention can be suitably used in various applications such as animplantable or circulation type hybrid artificial organ, materialproduction devices (bioreactors) by means of cells, and cell incubators(such as stem-cell amplifiers) for growing rare cells.

1. A cell-filled device of a modified cross-section hollow fibermembrane type, comprising hollow fiber membranes whose hollow portionsare filled with cells, characterized in that: the hollow fiber membraneshave modified cross sections; and a cell aggregate provided in each ofthe hollow portions has cells formed into two or more layers inarbitrary directions, provided that a distance from an arbitrary pointof the cell aggregate to the nearest inner wall of the hollow fibermembrane is less than 75 μm.
 2. A cell-filled device of a modifiedcross-section hollow fiber membrane type according to claim 1, whereinthe distance to the nearest inner wall of the hollow fiber membrane is50 μm or less.
 3. A cell-filled device of a modified cross-sectionhollow fiber membrane type according to claim 1, characterized in that across-section of the modified cross-section hollow fiber membrane is ina flat form.
 4. A cell-filled device of a modified cross-section hollowfiber membrane type according to claim 1, characterized in that a poresize of the hollow fiber membrane is 0.001 to 5 μm.
 5. A cell-filleddevice of a modified cross-section hollow fiber membrane type accordingto claim 4, wherein the pore size is 0.05 to 1 μm.
 6. A cell-filleddevice of a modified cross-section hollow fiber membrane type accordingto claim 1, characterized in that the hollow fiber membrane is made of asynthetic polymer having a contact angle of 70 degrees or less.
 7. Acell-filled device of a modified cross-section hollow fiber membranetype according to claim 6, wherein the synthetic polymer comprises athermoplastic resin.
 8. A cell-filled device of a modified cross-sectionhollow fiber membrane type according to claim 7, wherein thethermoplastic resin comprises a polyethylene-based resin.
 9. Acell-filled device of a modified cross-section hollow fiber membranetype according to claim 1, characterized in that at least an innersurface of the hollow fiber membrane contains a hydrophilic polymer. 10.A cell-filled device of a modified cross-section hollow fiber membranetype according to claim 1, characterized in that the cells comprisecells derived from an animal tissue.
 11. A cell-filled device of amodified cross-section hollow fiber membrane type according to claim 10,characterized in that the cells derived from an animal tissue compriseat least one kind of cell selected from the group consisting of cellsderived from a liver, cells derived from a spleen, stem and precursorcells thereof, and genetic recombinant cells.
 12. A cell-filled deviceof a modified cross-section hollow fiber membrane type according toclaim 11, characterized in that the cells derived from an animal tissuecomprise hepatic cells.
 13. A cell-filled device of a modifiedcross-section hollow fiber membrane type according to claim 10, whereinthe cells derived from an animal tissue comprise cells derived from ahuman organ.
 14. A cell-filled device, comprising hollow fiber membranesand cells, provided as the cell-filled device of a modifiedcross-section hollow fiber membrane type for implantation according toclaim 1, wherein each of the hollow portions contains a cell aggregateand both ends of each hollow fiber membrane are sealed.
 15. Acell-filled device of a modified cross-section hollow fiber membranetype for a hybrid artificial organ, which is one according to claim 1.16. A hybrid artificial organ, comprising at least one cell-filleddevice of a modified cross-section hollow fiber membrane type accordingto claim
 1. 17. A hybrid artificial organ, comprising at least onecell-filled device of a modified cross-section hollow fiber membranetype according to claim 1, being housed in a container having an inletand an outlet for a liquid to be treated, characterized in that aninside of a hollow of the cell-filled device of a modified cross-sectionhollow fiber membrane type is separated from an external of the hollowforming a communication path of the liquid to be treated.
 18. A methodof manufacturing a cell-filled device of a modified cross-section hollowfiber membrane type, comprising the steps of: a) obtaining a modifiedcross-section hollow fiber membrane; and b) injecting a cell suspensioninto a hollow of the hollow fiber membrane.
 19. A method ofmanufacturing a cell-filled device of a modified cross-section hollowfiber membrane type according to claim 18, further comprising the stepof producing the hollow fiber membrane using double annular spinningnozzle having a modified cross section.
 20. A method of manufacturing acell-filled device of a modified cross-section hollow fiber membranetype according to claim 18, further comprising the step of applying anexternal force for deformation to a hollow fiber membrane not having ashape of interest in an approximately vertical direction of its fiberaxis to obtain the modified cross-section hollow fiber membrane.
 21. Amethod of manufacturing a cell-filled device of a modified cross-sectionhollow fiber membrane type according to claim 18, further comprising thestep of drafting the hollow fiber membrane not having a shape ofinterest in the direction of its fiber axis while deforming the shape ofthe cross section to mold the membrane into the modified cross-sectionhollow fiber membrane.
 22. A method of manufacturing a cell-filleddevice of a modified cross-section hollow fiber membrane type accordingto claim 18, characterized in that the cross section of the modifiedcross-section hollow fiber membrane is in a flat form.
 23. A method ofmanufacturing a cell-filled device of a modified cross-section hollowfiber membrane type according to claim 18, characterized in that amaterial of the hollow fiber membrane comprises a thermoplastic resin.24. A method of manufacturing a cell-filled device of a modifiedcross-section hollow fiber membrane type according to claim 18,characterized in that injected cells comprise cells derived from ananimal tissue.
 25. A method of manufacturing a cell-filled device of amodified cross-section hollow fiber membrane type according to claim 24,characterized in that the cells derived from an animal tissue compriseat least one kind of cell selected from the group consisting of cellsderived from a liver, cells derived from a spleen, stem and precursorcells thereof, and genetic recombinant cells.
 26. A method ofmanufacturing a cell-filled device of a modified cross-section hollowfiber membrane type according to claim 25, characterized in that thecells derived from an animal tissue comprise hepatic cells.
 27. A methodof manufacturing a cell-filled device of a modified cross-section hollowfiber membrane type according to claim 24, characterized in that thecells derived from an animal tissue comprise cells derived from a humanorgan.
 28. A method of manufacturing a hybrid artificial organcomprising the method of manufacturing a cell-filled device according toclaim
 18. 29. A method of manufacturing a hybrid artificial organcharacterized in that: at least one modified cross-section hollow fibermembrane used in the method of manufacturing a cell-filled deviceaccording to claim 18 is housed in a container having an inlet and anoutlet for a liquid to be treated, and an injection opening for cells;and potting is performed such that an inside of a hollow is communicatedwith the injection opening for cells and separated from an externalportion of the hollow, followed by injecting cells into hollow portionsto form a cell aggregate.